In the field of culturing and detecting microorganisms, specialized culture bottles and machines for holding the culture bottles are typically used for detecting the presence of microorganisms in a test specimen. Bottles, such as those disclosed in U.S. Pat. Nos. 4,945,060; 5,094,955; and 5,162,229, herein incorporated by reference, have a culture medium and a sensor in the interior of the bottle that undergoes a detectable change due to the growth of microorganisms present in the bottle. The change in the sensor is monitored from outside the culture bottle through the transparent wall of the culture bottle, such as with a light emitter and detector as disclosed in, for example, U.S. Pat. Nos. 5,164,796 and 5,217,876, herein incorporated by reference. For most assays, the culture bottles should be agitated for best results. Clips, such as those disclosed in U.S. Pat. No. 5,074,505, herein incorporated by reference, can hold the culture bottles in place in the incubating machine during agitation.
The detection of pathogenic microorganisms in biological fluids should be performed in the shortest possible time, in particular in the case of septicemia for which the mortality remains high in spite of the broad range of antibiotics which are available to doctors. In order to increase sick individuals' chances of survival, practitioners often administer an antibiotic or mixture of antibiotics to the patients. It is, however, important to determine a suitable antibiotic therapy as soon as possible. Unfortunately, when a patient's bodily fluid sample is cultured to identify and isolate an infecting microorganism that might be present, antibiotics previously administered to the patient can interfere with the culturing process. Furthermore, medical samples may contain serum, plasma and lysed erythrocytes that may naturally contain microbial inhibitors. Industrial samples such as pharmaceuticals and foods may also contain antimicrobials or preservatives that inhibit the growth of microorganisms. Additionally, when culture media is prepared, autoclaving of the media at very high temperatures under pressure can result in the formation of by-products toxic to microorganisms. Removal or neutralization of these inhibitory or bactericidal substances is necessary to detect microbial contamination.
The use of resins and non-resinous adsorbents is well known and has been previously described for use in medical diagnostic procedures. In particular, these resins and non-resinous adsorbents have been shown useful in the removal of antibiotics and other antimicrobials from blood samples. The removal of these inhibitors in medical samples allows for recovery and faster detection of microorganisms so that microbial identification and accurate antibiotic susceptibility testing can be performed.
Melnick et al., U.S. Pat. No. 4,145,304, herein incorporated by reference, describes the use of synthetic anionic exchange and nonionic resins to remove antimicrobials, including antibiotics, from body fluids, thus allowing for recovery of pathogens using standard culture techniques. The resins described are coated with a nonionic detergent in order to selectively remove charged antibiotics while inhibiting adherence of bacteria to the resins. After treatment of the sample with the resin, the eluate is cultured in a growth media. The degree of binding of antibiotics by the resins is indicated to be dependent on the total exchange capacity, pore diameter, and surface area of the resin.
Waters, U.S. Pat. No. 4,632,902, herein incorporated by reference, describes an improvement over Melnick by incorporating ion exchange resins and non-functional adsorbent resins directly into the growth medium. Inhibitors removed include antibiotics administered to patients and naturally occurring inhibitors contained in serum, plasma, and lysed erythrocytes. The resins are not coated with a nonionic detergent or surfactant before use and the pore size of the resin is not critical.
Thorpe et al., U.S. Pat. Nos. 5,162,229 and 5,314,229, herein incorporated by reference, describe the use of resin and non-resinous adsorbents to neutralize, bind, or inhibit antimicrobial substances that may be present in a biological sample. The resin and non-resinous adsorbents described include, aluminum oxide, colloidal native hydrated aluminum silicates, crystalline hydrated alkali-aluminum silicates, silica, siliceous frustules, fragments of various species of diatoms, amorphous carbon, ion exchange resins, non-functional polymeric resin adsorbents, polystyrene resin cross-linked with divinyl benzene and combinations thereof.
Although synthetic resins and non-resinous adsorbents are known to remove inhibitory substances in cultures containing body fluids, these resins and non-resinous adsorbents may be ineffective in neutralizing or removing some types of antibiotics. For example, some β-lactams are commonly used in the treatment of sepsis and their presence in blood samples can interfere with the recovery, detection and identification of the microorganism responsible for the sepsis. In particular, resins and non-resinous adsorbents currently used in the art to remove antimicrobials from blood samples have proven largely ineffective in neutralizing carbapenems. It is therefore desirable to find other means for the neutralization or inhibition of antibiotics in body fluids and non-body fluid samples, such as foods and industrial products.
The present invention provides a means for the neutralization or inhibition of antimicrobials in test samples, while helping to retain the components of the medium necessary to recover and detect microorganisms in a rapid manner. By finding a means for the neutralization and/or inactivation of β-lactams (e.g., carbapenems) that previously could not be effectively neutralized or inactivated in a culture medium, the present invention solves a long-felt need in the art.